The mycobacterial species, Mycobacterium marinum, is a marine pathogen that infects poikilothermic animals such as fish and frogs and will also infect humans. M. marinum can form biofilms both in the laboratory and in the environment, but little is known about the molecular factors responsible for M. marinum biofilm formation. In this application, we describe the use of a flow-cell system for the study of M. marinum biofilm formation in situ. We will quantify biofilm formation and observe deposition of organisms upon a plastic matrix using both standard and fluorescence microscopy. We will further harvest M. marinum biofilms grown in flow cells for use in a differential fluorescence induction (DFI) system. In this way, we will be able to isolate promoters specifically induced during biofilm formation. In a complimentary approach to the DFI studies, we will isolate proteins from harvested M. marinum biofilms and the planktonic (free-floating) counterparts within the same culture and subject these proteins to 2-dimensional gel electrophoresis. This will allow the identification of mycobacterial proteins specific to biofilm formation. Promoters and proteins isolated will be compared to known M. marinum sequences as well as sequences available in other mycobacterial databases. We will, using these methods, be able to identify specific genes required for mycobacterial biofilm formation. In addition, we will be able to visualize the location of specific gene products within the biofilm using the same reporter assay system and confocal microscopy on the flow-cell-generated biofilm. These studies will lead to an understanding of the mechanisms by which mycobacteria form biofilms in nature and on artificial surfaces, and a more specific understanding of how biofilms can contribute to the pathogenesis of these organisms. [unreadable] [unreadable]